Platelet aggregation, activation and adhesion to endothelial cells and extracellular matrix proteins are crucial events in the development of atherosclerosis and arterial cardiovascular diseases. Platelet activation is initiated by stimulation of intracellular signaling cascades, including the p42 mitogen-activated protein kinase (MAPK) and p38 MAPK pathways, followed by major changes in the platelet cytoskeleton and expression and activation of platelet surface receptors, such as P-selectin (CD62P) and CD154.
Mural and intraplaque arterial thrombi are composed primarily of platelet aggregates and are physically associated with developing lesions. Activated platelets present in a platelet-rich arterial thrombi directly bind to vascular endothelial cells, smooth muscle cells, and other cells within an atheroma, via CD154/CD40 interactions. This binding induces inflammatory reactions that have the potential of initiating or hastening the development of atherosclerotic lesions, as detected by the release of inflammatory cytokines and the surface expression on endothelial cells of adhesion receptors and tissue factor. This pathway is also important for T cell-induced monocyte and endothelial cell procoagulant activity. It is believed that human clinical trials are currently underway, designed to study whether the administration of soluble CD154 can enhance immune response in certain patients.
Platelets co-localize with leukocytes at sites of hemorrhage, within atherosclerotic and postangioplasty restenotic lesions, and in areas of ischemia-reperfusion injury. This interaction between platelets and leukocytes illustrates the biological links between hemostatic/thrombotic and inflammatory responses. It has been suggested that inflammation and thrombosis are more intertwined in the processes of vascular disease than was previously appreciated.
The vascular endothelium influences the three classically interacting components of hemostasis: blood vessels, platelets, and the clotting and fibrinolytic systems of plasma. Additionally, the vascular endothelium plays an important role in inflammation and tissue repair. Two principal modes of endothelial behavior may be differentiated, best defined as an antithrombotic state and as a prothrombotic state. Under typical physiological conditions, the endothelium mediates vascular dilatation (formation of NO, PGI2, adenosine, hyperpolarizing factor), prevents platelet adhesion and activation (production of adenosine, NO and PGI2, removal of ADP), blocks thrombin formation (tissue factor pathway inhibitor, activation of protein C via thrombomodulin, activation of antithrombin III) and mitigates fibrin deposition (t- and scuplasminogen activator production). Adhesion and transmigration of inflammatory leukocytes are attenuated, e.g., by NO and IL-10, and oxygen radicals are efficiently scavenged (urate, NO, glutathione, SOD; see, e.g., Becker, B F et al., Z. Kardiol 2000; 89(3):160-167).
When the endothelium is physically disrupted or functionally perturbed by postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension, then opposing actions pertain. This prothrombotic, proinflammatory state is characterized by vaso-constriction, platelet and leukocyte activation and adhesion (externalization, expression and upregulation of von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, TNF alpha, etc.), promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall (expression of tissue factor, PAI-1, phosphatidyl serine, etc.) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelets to endothelial, monocyte and B-cell CD154.
The role of endothelial inflammation in the progression of atherosclerosis has been shown (Libby, P et al., in Atherosclerosis and Coronary Artery Disease, Fuster V, et al, eds. Lippincot-Raven, Philadelphia. 585-594 (1996); R. Ross, New Eng. J. Med.; 340:115-126 (1999)), and this has lead to efforts directed towards understanding the inflammatory factors responsible for lesion progression and identifying agents to regulate the inflammatory response.
CD40 is a 45-50 kD transmembrane glycoprotein found on many vascular cells (Hollenbough, D, J. Exp. Med. 282:33 (1995)), including B lymphocyte lineage cells. CD40 is a member of the TNF receptor family and has homology to the receptors for nerve growth factor, TNF-alpha, Fas and CD30. CD40 has been shown to play an important role in B cell differentiation and activation. Its primary role on immune cells is to enhance their activation and hence their production of cytokines and immunomodulatory molecules. Recently, CD40 has also been detected on human fibroblasts. An emerging view of the fibroblast is that it is far more than a structural cell, being capable of intimate interaction with cells of the immune system. In fibroblasts from several tissues, the engagement of CD40 with its ligand resulted in the secretion of proinflammatory molecules such as interleukin-6 (IL-6) and IL-8.
The ligand for CD40 is CD154 (also known as CD40 Ligand, CD40L and gp 39). CD154 is a membrane protein in the TNF family, and is a potent proinflammatory factor originally identified in CD4+ T lymphocytes. CD154 was identified as a factor responsible for immunoglobulin isotype switching (Gordon, J, Eur. J. Immunol. 17:1535 (1987)). As expressed on activated T cells, it is a type II membrane protein (N-terminus intracellular and C-terminus extracellular) cells. The human CD154 protein is 261 residues long and has a single N-linked carbohydrate moiety.
Previous studies have established that CD154 is rapidly released after T cell activation (Graf, D, Eur. J. Immunol. 25:1749 (1995)), creating an 18 kDa hydrolytic product capable of inducing B cell proliferation (Pietravalle, F J. Biol. Chem., 271:5965 (1996)) and an inflammatory response in vascular cells (Schonbeck, 1997). Mutations in the CD154 gene are associated with a rare immunodeficiency state, X-linked hyper IgM syndrome (XLHIGM).
Antibodies to CD154 have been shown to suppress T cell and antibody mediated immune responses in a number of experimental systems. These include inhibition of graft rejection and blocking autoirunmune disorders (Durie, F H et al., Science, 261:1328 (1993)). The combined use of anti-CD154 antibodies and CD28 blockers (i.e., CTLA-4Ig) has been shown to be effective in blocking graft rejection in both murine and rhesus transplant models (Larsen, C P et al., Nature, 381: 434 (1996); Kirk, A D, Proc. Natl. Acad. Sci. (USA), 94:8789(1997)). Other studies have shown that the use ofanti-CD154 antibody as a single agent in rhesus kidney allografts has shown that this treatment is remarkably efficacious (Kirk, A D et al., Nature Medicine 5: 686.(1999)). Clinical studies are believed to be underway, using anti-CD154 humanized antibodies for treatment of lupus or other autoimmune disease.
CD154 is also known to be a key mediator of atherosclerotic lesion progression. This role of CD154 in atherosclerosis has been established by studies showing that CD154 antibodies (Mach, F, Nature, 394:200 (1998)) or CD154 gene targeting (Lutgens, E, Nature Medicine, 5:1313 (1999) reduce atherosclerotic lesion development in the apo E-/- mouse.
Aukrust, et al. (Circulation; 100(6):614-620 (1999)), showed that a soluble form of CD154 was generated in patients with acute coronary thrombotic syndromes (eg., with unstable angina, or undergoing angioplasty and that the soluble CD154 was inflammatory to peripheral blood monocytes. From these observations, it was postulated that the soluble CD154 thus generated may trigger and/or propagate acute coronary syndromes.
CD154 is now known to reside in many cells within the vasculature, including platelets (Mach, F., Proc. Natl. Acad. Sci. (USA), 94:1931 (1997)); Banchereau, Ann Rev. Irrununol. 12:881 (1994)). Henn, et al., who showed that while CD154 was not exposed on the surface of unstimulated, discoid platelets, it rapidly became exposed on platelets during thrombin-induced aggregation, established the platelet location of CD154 in a pioneering study.
Phipps et al. (The Lancet, 357:2023-2024 (2001)) further elucidated that platelet soluble CD40 ligand is the cause of febrile responses and that keeping the release of CD40 ligand to a minimum or removing free CD40 ligand before transfusion may effectively reduce adverse events after platelet transfusion.
Eliopoulos et al. (Molecular Cell Biology, 20(15):5503-5515 (2000)) found that CD154 induced apoptosis through CD40 in carcinoma cells and identified a proapoptotic mechanism which depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.
There exists a need for therapeutic agents that can reduce the contributions made by platelets, via CD154/CD40 interactions, to atherosclerosis and other acute coronary syndromes. Furthermore, in view of the pro-inflammatory role of CD154, and given the potential therapeutic results of inhibiting the activity and effects of CD154, it would be desirable to have high affinity and high specificity inhibitors of this molecule.
Therapeutic agents directed to the prevention or reduction of platelet aggregation are known. For example, it has been suggested that administering a metalloproteinase inhibitor can reduce or block platelet aggregation. Certain other platelet blocking agents are directed against the platelet glycoprotein IIb-IIIa receptor that is involved in platelet aggregation. Large, randomized clinical trials have established that the three parenteral GPIIb-IIIa antagonists (abciximab, aggrastat and eptifibatide (INTEGRILIN®; Millennium Pharmaceuticals, Inc.)) reduce the incidence of acute coronary thrombosis in the settings of percutaneous interventions and unstable angina (Topol, E., Circulation, 97, 211 (1998)). One additional, unexpected benefit of this class of drugs is that they also augment the anticoagulant activity of heparin. It has been suggested that the drugs achieve this benefit, perhaps because they limit the expression of prothrombinase found on aggregated platelets and/or because they inhibit the binding of prothrombin to GP IIb-IIIa. Another unexpected benefit of this class of drugs is that they augment clot lysis by thrombolytics, perhaps because they inhibit platelet aggregation created by the prothrombotic environment created by fibrinolysis.
While these agents are beneficial to certain patients in these settings, further progress must be made toward improving treatment outcomes. Angioplasty, for example, has been shown to benefit patients, however the procedure is not without its adverse effects and complications. Recent clinical data from patients with vascular disease suggest that outcomes are not as good when inflammation is elevated as they are in patients without signs of inflammation. Despite a long-felt need to understand and discover methods for regulating thrombosis and inflammation, the complexity of the cellular interactions has complicated the development of completely satisfactory, safe and efficacious products and processes for regulating hemostatic function. As such, there remains a need for products and processes that permit the implementation of predictable controls of vascular inflammation and thrombosis, thus enabling the treatment of various diseases that are caused by undesired cellular function.